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1.
Colleen J. Mate Susanne von Caemmerer John R. Evans Graham S. Hudson T. John Andrews 《Planta》1996,198(4):604-613
Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) activase were used to examine the relationship between CO2-assimilation rate, Rubisco carbamylation and activase content. Plants used were those members of the r1 progeny of a primary transformant with two independent T-DNA inserts that could be grown without CO2 supplementation. These plants had from < 1% to 20% of the activase content of control plants. Severe suppression of activase to amounts below 5% of those present in the controls was required before reductions in CO2-assimilation rate and Rubisco carbamylation were observed, indicating that one activase tetramer is able to service as many as 200 Rubisco hexadecamers and maintain wild-type carbamylation levels in vivo. The reduction in CO2-assimilation rate was correlated with the reduction in Rubisco carbamylation. The anti-activase plants had similar ribulose-1,5-bisphosphate pool sizes but reduced 3-phosphoglycerate pool sizes compared to those of control plants. Stomatal conductance was not affected by reduced activase content or CO2-assimilation rate. A mathematical model of activase action is used to explain the observed hyperbolic dependence of Rubisco carbamylation on activase content.Abbreviations CA1P
2-carboxyarabinitol-1-phosphate
- Pipa
intercellular, ambient partial pressure of CO2
- PGA
3-phospho-glycerate
- Rubisco
ribulose-1,5-bisphosphate carboxylase/oxygenase
- RuBP
ribulose-1,5-bisphosphate
- SSU
small subunit of Rubisco 相似文献
2.
A simple approach to determine CO2/O2 specificity factor () of ribulose 1,5-bisphosphate carboxylase/oxygenase is described. The assay measures the amount of CO2 fixation at varying [CO2]/[O2] ratios after complete consumption of ribulose 1,5-bisphosphate (RuBP). Carbon dioxide fixation catalyzed by the carboxylase was monitored by directly measuring the moles of 14CO2 incorporated into 3-phosphoglycerate (PGA). This measurement at different [CO2]/[O2] ratios is used to determine graphically by several different linear plots the total RuBP consumed by the two activities and the CO2/O2 specificity factor. The assay can be used to measure the amounts of products of the carboxylase and oxygenase reactions and to determine the concentration of the substrate RuBP converted to an endpoint amount of PGA and phosphoglycolate. The assay was found to be suitable for all [CO2]/[O2] ratios examined, ranging from 14 to 215 micromolar CO2 (provided as 1–16 mM NaHCO3) and 614 micromolar O2 provided as 50% O2. The procedure described is extremely rapid and sensitive. Specificity factors for enzymes of highly divergent values are in good agreement with previously published data.Abbreviations HEPPS
N-(2-hydroxyethyl)piperazine-N-(3-propanesulfonic acid)
- L
large subunit of rubisco
- PGA
3-phosphoglyceric acid
- rubisco
ribulose 1,5-bisphosphate carboxylase/oxygenase
- RuBP
d-ribulose 1,5-bisphosphate
- S
small subunit of rubisco
- XuBP
d-xylulose 1,5-bisphosphate 相似文献
3.
Ian E. Woodrow 《Photosynthesis research》1994,39(3):401-412
A range of studies of C3 plants have shown that there is a change in both the carbon flux and the pattern of nitrogen allocation when plants are grown under enhanced CO2. This paper examines evidence that allocation of nitrogen both to and within the photosynthetic system is optimised with respect to the carbon flux. A model is developed which predicts the optimal relative allocation of nitrogen to key enzymes of the photosynthetic system as a function of CO2 concentration. It is shown that evidence from flux control analysis is broadly consistent with this model, although at high nitrogen and under certain conditions at low nitrogen experimental data are not consistent with the model. Acclimation to enhanced CO2 is also assessed in terms of resource allocation between photosynthate sources and sinks. A means of assessing the optimisation of this source-sink allocation is proposed, and several studies are examined within this framework. It is concluded that C3 plants probably possess the genetic feedback mechanisms required to efficiently smooth out any imbalance within the photosynthetic system caused by a rise in atmospheric CO2.Abbreviations A
net rate of CO2 assimilation
-
c
i
intercellular CO2 concentration
- CR
A
flux control coefficient for Rubisco with respect to flux A
- FBPase
fructose 1,6-bisphosphatase
- kapp
apparent catalytic rate constant
- PCO
photorespiratory carbon oxidation
- PCR
photosynthetic carbon reduction
- PPFD
photosynthetically active photon flux density
- Rubisco
ribulose 1,5-bisphosphate carboxylase/oxygenase
- RuBP
ribulose 1,5-bisphosphate
- Ru5P
ribulose 5-phosphate
- SBPase
sedoheptulose 1,7-bisphosphatase 相似文献
4.
Determination of Apparent K(m) Values for Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Using the Spectrophotometric Assay of Rubisco Activity 总被引:3,自引:0,他引:3
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The spectrophotometric assay for ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) was used to determine the rate of increase in Rubisco activity over time in the presence or absence of Rubisco activase. Polynomial approximations to the raw data were used to smooth out minor fluctuations in the spectrophotometer readings, and Rubisco activase activity was expressed as nanomoles of activated Rubisco per minute. This assay was used to examine the effects of CO2 and the inactive-Rubisco:ribulose 1,5-bisphosphate complex (ER) on the activase-catalyzed activation reaction. Double-reciprocal plots of activase activity and ER at several concentrations of CO2 were consistent with two-substrate Michaelis-Menton kinetics, and the apparent Km (CO2) and Km(ER) were determined to be 53 and 2.7 micromolar, respectively. These data do not prove that ER and CO2 are substrates for the reaction catalyzed by activase, but they may be important to our understanding of the activation process in vivo. The implications of these data and their relation to previously published data on the effects of ER and CO2 on activase are discussed. 相似文献
5.
Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans
Synechococcus PCC 6301) was used to generate novel enzymes in Escherichia coli. Residues in C-terminal loop 6 of the / barrel structure of the large subunit were changed. Replacement of valine 331 with alanine caused a 90% reduction in V
max but did not alter the enzyme's relative specificity towards either of its gaseous substrates, CO2 and O2. However replacement of alanine 340 with glutamate decreased the enzyme's specificity for CO2 but had no significant effect on either the K
m for ribulose-1,5-bisphosphate or CO2 or on V
max. In contrast replacing a small cassette of residues 338-341 produced a small increase in the specificity factor.Abbreviations Rubisco
ribulose-1,5-bisphosphate carboxylase/oxygenase
- RuBP
ribulose-1,5-bisphosphate
- CABP
2-carbox-yarabinitol-1,5-bisphosphate
We thank Karen Moore for the statistical analysis of the specificity factors. We acknowledge helpful discussions with Jim Pitts and Richard Pickersgill. This work was aided by the invaluable technical assistance of Iain Major. 相似文献
6.
Zhu Genhai Bohnert Hans J. Jensen Richard G. Wildner Günter F. 《Photosynthesis research》1998,55(1):67-74
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (EC 4.1.1.39) not only catalyzes carboxylation and oxygenation of ribulose-1,5-bisphosphate (RuBP), but it can also act either as an epimerase or isomerase converting RuBP into xylulose-1,5-bisphosphate (XuBP) or 3-ketoarabinitol-1,5-bisphosphate (KABP), respectively, a process called misfire. XuBP is formed as a result of misprotonation at C3 of the RuBP-enediol. It is released from Rubisco active sites and accumulates in the reaction mixture. Increasing the amounts of CO2 or O2 decreases XuBP production. However, KABP synthesis, which has been proposed to be only a product due to C2 misprotonation of the RuBP-endiol, is dependent upon the presence of O2. KABP remains tightly bound to Rubisco active sites after its formation, causing the loss of Rubisco activity (fallover). The results suggest that the non-stabilized form of the peroxy-intermediate in the oxygenase reaction can be converted in a backreaction to KABP and molecular oxygen. The stabilization of the peroxy-intermediate due to the presence of Mn2+ instead of Mg2+ eliminates the formation of KABP. 相似文献
7.
Gas exchange and contents of photosynthetic intermediates of leaves of Arbutus unedo L. were determined with the aim of recognizing the mechanisms of inhibition that were responsible for the midday depression of photosynthesis following exposure to dry air, and the decline in photosynthetic capacity following application of abscisic acid (ABA). Rapidly killed (<0.1 s) leaf samples were taken when gas analysis showed reduced CO2 assimilation. Determination of the contents of 3-phosphoglyceric acid (PGA), ribulose 1,5-bisphosphate (RuBP), triose phosphates, fructose 1,6-bisphosphate and hexose phosphates in the samples showed that significant variation occurred only in the level of PGA. As a result, the ratio PGA/RuBP decreased with increasing inhibition of photosynthesis, particularly when application of ABA had been the cause. A comparison of metabolite patterns did not bring out qualitative differences that would have indicated that effects of ABA and of dry air had been caused by separate mechanisms. Depression of photosynthesis occurred in the presence of sufficient RuBP which indicated that the carboxylation reaction of the carbon-reduction-cycle was inhibited after application of ABA or exposure to dry air.Abbreviations and symbols ABA
abscisic acid
-
C
a
partial pressure of CO2 in the ambient air
-
C
i
partial pressure of CO2 in the intercellular spaces
- I
quantum flux
- PGA
3-phosphoglyceric acid
- RuBP
ribulose 1,5-bisphosphate
-
I
L
leaf temperature
- w
water-vapor pressure difference between leaf and air 相似文献
8.
A model of the C
3 photosynthetic system is developed which describes the sensitivity of the steadystate rate of carbon dioxide assimilation to changes in the activity of several enzymes of the system. The model requires measurements of the steady-state rate of carbon dioxide assimilation, the concentrations of several intermediates in the photosynthetic system, and the concentration of the active site of ribulose 1,5-bisphosphate carboxyalse/oxygenase (Rubisco). It is shown that in sunflowers (Helianthus annuus L.) at photon flux densities that are largely saturating for the rate of photosynthesis, the steady-stete rate of carbon dioxide assimilation is most sensitive to Rubisco activity and, to a lesser degree, to the activities of the stromal fructose, 6-bisphosphatase and the enzymes catalysing sucrose synthesis. The activities of sedoheptulose 1,7-bisphosphatase, ribulose 5-phosphate kinase, ATP synthase and the ADP-glucose pyrophosphorylase are calculated to have a negligible effect on the flux under the high-light conditions. The utility of this analysis in developing simpler models of photosynthesis is also discussed.Abbreviations
c
i
intercellular CO2 concentration
-
C
infP
supJ
control coefficient for enzyme P with respect to flux J
- DHAP
dihydroxyacetonephosphate
- E4P
erythrose 4-phosphate
- F6P
fructose 6-phosphate
- FBP
fructose 1,6-bisphosphate
- FBPase
fructose 1,6-bisphosphatase
- G3P
glyceraldehyde 3-phosphate
- G1P
glucose 1-phosphate
- G6P
glucose 6-phosphate
- Pi
inorganic phosphate
- PCR
photosynthetic carbon reduction
- PGA
3-phosphoglyceric acid
- PPFD
photosynthetically active photon flux density
-
R
n
J
response coefficient for effector n with respect to flux J
- R5P
ribose 5-phosphate
- Rubisco
ribulose 1,5-bisphosphate carboxylase/oxygenase
- Ru5P
ribulose 5-phosphate
- RuBP
ribulose 1,5-bisphosphate
- S7P
sedoheptulose 7-phosphate
- SBP
sedoheptulose 1,7-bisphosphate
- SBPase
sedoheptulose 1,7-bisphosphatase
- SPS
sucrose-phosphate synthase
- Xu5P
xylulose 5-phosphate
-
n
P
elasticity coefficient for effector n with respect to the catalytic velocity of enzyme P
This research was funded by an Australian Research Council grant to I.E.W. and was undertaken during a visity by K.A.M. to the James Cook University of North Queensland. The expert help of Glenys Hanley and Mick Kelly is greatly appreciated. 相似文献
9.
Transgenic tobacco (Nicotiana tabacum L.) plants transformed with antisense rbcS to produce a series of plants with a progressive decrease in the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been used to investigate the contribution of Rubsico to the control of photosynthesis at different irradiance, CO2 concentrations and vapour-pressure deficits. Assimilation rates, transpiration, the internal CO2 concentration and chlorophyll fluorescence were measured in each plant. (i) The flux-control coefficient of Rubisco was estimated from the slope of the plot of Rubisco content versus assimilation rate. The flux-control coefficient had a value of 0.8 or more in high irradiance, (1050 mol·m–2·s–1), low-vapour pressure deficit (4 mbar) and ambient CO2 (350 bar). Control was marginal in enhanced CO2 (450 bar) or low light (310 mol·m–2·s–1) and was also decreased at high vapour-pressure deficit (17 mbar). No control was exerted in 5% CO2. (ii) The flux-control coefficients of Rubisco were compared with the fractional demand placed on the calculated available Rubisco capacity. Only a marginal control on photosynthetic flux is exerted by Rubisco until over 50% of the available capacity is being used. Control increases as utilisation rises to 80%, and approaches unity (i.e. strict limitation) when more than 80% of the available capacity is being used. (iii) In low light, plants with reduced Rubisco have very high energy-dependent quenching of chlorophyll fluorescence (qE) and a decreased apparent quantum yield. It is argued that Rubisco still exerts marginal control in these conditions because decreased Rubisco leads to increased thylakoid energisation and high-energy dependent dissipation of light energy, and lower light-harvesting efficiency. (iv) The flux-control coefficient of stomata for photosynthesis was calculated from the flux-control coefficient of Rubisco and the internal CO2 concentration, by applying the connectivity theorem. Control by the stomata varies between zero and about 0.25. It is increased by increased irradiance, decreased CO2 or decreased vapour-pressure deficit. (v) Photosynthetic oscillations in saturating irradiance and CO2 are suppressed in decreased-activity transformants before the steady-state rate of photosynthesis is affected. This provides direct evidence that these oscillations reveal the presence of excess Rubisco. (vi) Comparison of the flux-control coefficients of Rubisco with mechanistic models of photosynthesis provides direct support for the reliability of these models in conditions where Rubisco has a flux-control coefficient approach unity (i.e. limits photosynthesis), but also indicates that these models are less useful in conditions where control is shared between Rubisco and other components of the photosynthetic apparatus.Abbreviations A
assimilation rate
- Ci
intercellular CO2 concentration in the leaf
- CR
flux-control coefficient of Rubisco for photosynthesis
- qE
high-energy-state-dependent quenching of chlorophyll fluorescence
- QA
primary acceptor of PSII
-
rbc S
gene for the nuclear-encoded small subunit of Rubisco
- Rubisco
ribulose-1,5-bisphosphate carboxylase-oxygenase
- Ru1,5bisP
ribulose-1,5-bisphosphate
- VPD
vapour-pressure deficit 相似文献
10.
Howard Griffiths Mark S. J. Broadmeadow Anne M. Borland Clive S. Hetherington 《Planta》1990,181(4):604-610
Short-term measurements of instantaneous carbon-isotope discrimination have been determined from mass-spectrometric analyses of CO2 collected online during gas exchange for the epiphytic bromeliad Tillandsia utriculata L. Using this technique, the isotopic signature of CO2 exchange for each phase of Crassulacean acid metabolism (CAM) has been characterised. During night-time fixation of CO2 (Phase I), discrimination () ranged from 4.4 to 6.6, equivalent to an effective carbon-isotope ratio (13C) of –12.3 to –14.5 versus Pee Dee Belemnite (PDB). These values reflected the gross photosynthetic balance between net CO2 uptake and refixation of respiratory CO2, characteristic of CAM in the Bromeliaceae. When for the relative proportion of external (p
a
) and internal (p
i) CO2 is taken into account, calculated p
i/p
a decreased during the later part of the dark period from 0.68 to 0.48. Measurements of during Phase II, early in the light period, showed the transition between C4 and C3 pathways, with carboxylation being increasingly dominated by ribulose bisphosphate carboxylase (Rubisco) as increased from 10.5 to 21.2 During decarboxylation in the light period (Phase III), CO2 leaked out of the leaf and the inherent discrimination of Rubisco was expressed. The value of calculated from on-line measurements (64.4) showed that the CO2 lost was considerably enriched in 13C, and this was confirmed by direct analysis of the CO2 diffusing out into a CO2-free atmosphere (
13C = + 51.6 versus PDB). Instantaneous discrimination was characteristic of the C3 pathway during Phase IV (late in the light period), but a reduction in showed an increasing contribution from phosphoenolpyruvate carboxylase. The results from this non-invasive technique confirm the observations that double carboxylation involving both phosphoenolpyruvate carboxylase and Rubisco occurs during the transient phases of CAM (II and IV) in the light period.Abbreviations and Symbols CAM
Crassulacean acid metabolism
-
H+
(dawn-dusk) variation in titratable acidity
-
13C
carbonisotope ratio of plant organic material, relative to Pee Dee Belemnite (vs. PDB)
-
discrimination against 13CO2,
-
p
i, p
a
internal, external partial pressures of CO2
- Rubisco
ribulose1,5-bisphosphate carboxylase
- PAR
photosynthetically active radiation
- PEPCase
phosphoenolpyruvate carboxylase
We are grateful for financial support in respect of research grants (GR3/5360, GR3/6419) and a studentship awarded by the Natural Environment Research Council, UK. 相似文献
11.
Phosphorus-deficient spinach plants were grown by transferring them to nutrient solutions without PO4. Photosynthetic rates were measured at a range of intercellular CO2 partial pressures from 50–500 bar and then the leaves were freeze-clamped in situ to measure ribulose bisphosphate carboxylase (Rubisco) activity and metabolite concentrations. Compared with control leaves, deficient leaves had significantly lower photosynthetic rates, percentage activation of Rubisco, and amounts of ribulose bisphosphate and 3-phosphoglycerate at all CO2 partial pressures. After feeding 10 mM PO4 to the petioles of detached deficient leaves, all these measurements increased within 2 hours. At atmospheric CO2 partial pressure the photosynthetic rate was stimulated in 19 mbar O2 compared with 200 mbar. At higher CO2 partial pressures this stimulation was less but the percentage stimulation in deficient leaves was no different from controls in either CO2 partial pressure. It was concluded that phosphorus deficiency affects both Rubisco activity and the capacity for ribulose bisphosphate regeneration, and possible causes are discussed.Abbreviations A
CO2 assimilation rate
- Ci
intercellular CO2 partial pressure
- PGA
3-phosphoglycerate
- RuP2
ribulose 1,5-bisphosphate
- Rubisco
RuP2 carboxylase/oxygenase 相似文献
12.
A heterotrophic cotton (Gossypium hirsutum L. cv. Stoneville 825) cell suspension culture was adapted to grow photoautotrophically. After two years in continuous photoautotrophic culture at 5% CO2 (balance air), the maximum growth rate of the photoautotrophic cell line was a 400% fresh weight increase in eight days. The Chl concentration was approximately 500 g per g fresh weight.Elevated CO2 (1%–5%) was required for culture growth, while the ambient air of the culture room (600 to 700 ul CO2 1–1) or darkness were lethal. The cell line had no net photosynthesis at 350 ul 1–1 CO2, 2% O2, and dark respiration ranged from 29 to 44 mol CO2 mg–1 Chl h–1. Photosynthesis was inhibited by O2. The approximate 1:1 ratio of ribulose 1,5-bisphosphate carboxylase (RuBPcase) to phosphoenolpyruvate carboxylase (PEPcase) (normally about 6:1 in mature leaves of C3 plants) was due to low RuBPcase activity relative to that of C3 leaves, not to high PEPcase activity. The PEPcase activity per unit Chl in the cell line was identical to that of spinach leaves, while the RuBPcase activity was only 15% of the spinach leaf RuBPcase activity. RuBPcase activity in the photoautotrophic cells was not limited by a lack of activation in vivo, since the enzyme in a rapidly prepared cell extract was 73% activated. No evidence of enzyme inactivation by secondary compounds in the cells was found as can be found with cotton leaves. Low RuBPcase activity and high respiration rates are most likely important factors in the low photosynthetic efficiency of the cells at ambient CO2.Abbreviations Chl
chlorophyll
- COT
heterotrophic cotton cell line
- COT-P
photoautotrophic cotton cell line
- DCMU
3-(3,4-dichlorophenyl)-1,1-dimethylurea
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- Rubisco
ribulose 1,5-bisphosphate carboxylase/oxygenase
- RuBP
ribulose 1,5-bisphosphate
- RuBPcase
RuBP carboxylase
- PEP
phosphoenolpyruvate
- PEPcase
phosphoenolpyruvate carboxylase
- MX
Murashige and Skoog medium with 0.4 mg 1–1 2,4-D
- KT
photomixotrophic medium with 1% sucrose
- KTo
KT medium with no carbohydrate
- KTPo
KTo medium supplemented with 0.3 M Picloram
- CER
CO2 exchange rate
- PCER
CO2 exchange rate in the light 相似文献
13.
The substrate specificity factor, V
cKo/VoKc, of spinach (Spinacia oleracea L.) ribulose 1,5-bisphosphate carboxylase/oxygenase was determined at ribulosebisphosphate concentrations between 0.63 and 200 M, at pH values between 7.4 and 8.9, and at temperatures in the range of 5° C to 40° C. The CO2/O2 specificity was the same at all ribulosebisphosphate concentrations and largely independent of pH. With increasing temperature, the specificity decreased from values of about 160 at 5° C to about 50 at 40° C. The primary effects of temperature were on K
c [Km(CO2)] and V
c [Vmax (CO2)], which increased by factors of about 10 and 20, respectively, over the temperature range examined. In contrast, K
o [Ki (O2)] was unchanged and V
o [Vmax (O2)] increased by a factor of 5 over these temperatures. The CO2 compensation concentrations () were calculated from specificity values obtained at temperatures between 5° C and 40° C, and were compared with literature values of . Quantitative agreement was found for the calculated and measured values. The observations reported here indicate that the temperature response of ribulose 1,5-bisphosphate carboxylase/oxygenase kinetic parameters accounts for two-thirds of the temperature dependence of the photorespiration/photosynthesis ratio in C3 plants, with the remaining one-third the consequence of differential temperature effects on the solubilities of CO2 and O2.Abbreviations RuBPC/O(ase)
ribulose 1,5-bisphosphate carboxylase/oxygenase
- RuBP
ribulose 1,5-bisphosphate
-
CO2 compensation concentration 相似文献
14.
In the cyanobacterium Synechococcus UTEX 625, the extent of expression of carboxysomes appeared dependent on the level of inorganic carbon (CO2+HCO
inf3
sup-
) in the growth medium. In cells grown under 5% CO2 and in those bubbled with air, carboxysomes were present in low numbers (<2 · longitudinal section-1) and were distributed in an apparently random manner throughout the centroplasm. In contrast, cells grown in standing culture and those bubbled with 30 l CO2 · 1-1 possessed many carboxysomes (>8 · longitudinal section-1). Moreover, carboxysomes in these cells were usually positioned near the cell periphery, aligned along the interface between the centroplasm and the photosynthetic thylakoids. This arrangement of carboxysomes coincided with the full induction of the HCO
inf3
sup-
transport system that is involved in concentrating inorganic carbon within the cells for subsequent use in photosynthesis. Immunolocalization studies indicate that the Calvin cycle enzyme ribulose bisphosphate carboxylase was predominantly carboxysome-localized, regardless of the inorganic carbon concentration of the growth medium, while phosphoribulokinase was confined to the thylakoid region. It is postulated that the peripheral arrangement of carboxysomes may provide for more efficient photosynthetic utilization of the internal inorganic carbon pool in cells from cultures where carbon resources are limiting.Abbreviations Chl
chlorophyll
- DIC
dissolved inorganic carbon (CO2+HCO
inf3
sup-
+CO
inf3
sup2-
)
- PRK
phosphoribulokinase
- RuBP
ribulose 1,5-bisphosphate
- Rubisco LS
large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase 相似文献
15.
The kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase in vivo inferred from measurements of photosynthesis in leaves of transgenic tobacco 总被引:20,自引:0,他引:20
Transgenic tobacco (Nicotiana tabacum L. cv. W38) with an antisense gene directed against the mRNA of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit was used to determine the kinetic properties of Rubisco in vivo. The leaves of these plants contained only 34% as much Rubisco as those of the wild type, but other photosynthetic components were not significantly affected. Consequently, the rate of CO2 assimilation by the antisense plants was limited by Rubisco activity over a wide range of CO2 partial pressures. Unlike in the wild-type leaves, where the rate of regeneration of ribulose bisphosphate limited CO2 assimilation at intercellular partial pressures above 400 ubar, photosynthesis in the leaves of the antisense plants responded hyperbolically to CO2, allowing the kinetic parameters of Rubisco in vivo to be inferred. We calculated a maximal catalytic turnover rate, kcat, of 3.5+0.2 mol CO2·(mol sites)–1·s–1 at 25° C in vivo. By comparison, we measured a value of 2.9 mol CO2·(mol sites)–1·–1 in vitro with leaf extracts. To estimate the Michaelis-Menten constants for CO2 and O2, the rate of CO2 assimilation was measured at 25° C at different intercellular partial pressures of CO2 and O2. These measurements were combined with carbon-isotope analysis (13C/12C) of CO2 in the air passing over the leaf to estimate the conductance for transfer of CO2 from the substomatal cavities to the sites of carboxylation (0.3 mol·m–2·s–1·bar–1) and thus the partial pressure of CO2 at the sites of carboxylation. The calculated Michaelis-Menten constants for CO2 and O2 were 259 ±57 bar (8.6±1.9M) and 179 mbar (226 M), respectively, and the effective Michaelis-Menten constant for CO2 in 200 mbar O2 was 549 bar (18.3 M). From measurements of the photocompensation point (* = 38.6 ubar) we estimated Rubisco's relative specificity for CO2, as opposed to O2 to be 97.5 in vivo. These values were dependent on the size of the estimated CO2-transfer conductance.Abbreviations and Symbols A
CO2-assimilation rate
- gw
conductance for CO2 transfer from the substomatal cavities to the sites of carboxylation
- Kc, Ko
Michaelis-Menten constants for carboxylation, oxygenation of Rubisco
- kcat
Vcmax/[active site]
- O
partial pressure of O2 at the site of carboxylation
- pc
partial pressure of CO2 at the site of carboxylation
- pi
intercellular CO2 partial pressure
- Rd
day respiration (non-photorespiratory CO2 evolution)
- Rubisco
ribulose 1,5-bisphosphate carboxylase/oxygenase
- RuBP
ribulose-1,5-bisphosphate
- Sc/o
relative specificity factor for Rubisco
- SSu
small subunit of Rubisco
- Vcmax, Vomax
maximum rates of Rubisco carboxylation, oxygenation
- *
partial pressure of CO2 in the chloroplast at which photorespiratory CO2 evolution equals the rate of carboxylation 相似文献
16.
The short-term, in-vivo response to elevated CO2 of ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39) activity, and the pool sizes of ribulose 1,5-bisphosphate, 3-phosphoglyceric acid, triose phosphates, fructose 1,6-bisphosphate, glucose 6-phosphate and fructose 6-phosphate in bean were studied. Increasing CO2 from an ambient partial pressure of 360–1600 bar induced a substantial deactivation of RuBPCase at both saturating and subsaturating photon flux densities. Activation of RuBPCase declined for 30 min following the CO2 increase. However, the rate of photosynthesis re-equilibrated within 6 min of the switch to high CO2, indicating that RuBPCase activity did not limit photosynthesis at high CO2. Following a return to low CO2, RuBPCase activation increased to control levels within 10 min. The photosynthetic rate fell immediately after the return to low CO2, and then increased in parallel with the increase in RuBPCase activation to the initial rate observed prior to the CO2 increase. This indicated that RuBPCase activity limited photosynthesis while RuBPCase activation increased. Metabolite pools were temporarily affected during the first 10 min after either a CO2 increase or decrease. However, they returned to their original level as the change in the activation state of RuBPCase neared completion. This result indicates that one role for changes in the activation state of RuBPCase is to regulate the pool sizes of photosynthetic intermediates.Abbreviations and symbols
A
net CO2 assimilation rate
- Ca
ambient CO2 partial pressure
- Ci
intercellular CO2 partial pressure
- CABP
2-carboxyarabinitol 1,5-bisphosphate
- kcat
catalytic turnover rate per RuBPCase molecule
- PFD
photon flux density (400 to 700 nm on an area basis)
- PGA
3-phosphoglyceric acid
- Pi
orthophosphate
- RuBP
ribulose 1,5-bisphosphate
- RuBPCase
ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) 相似文献
17.
W. P. Quick U. Schurr R. Scheibe E.-D. Schulze S. R. Rodermel L. Bogorad M. Stitt 《Planta》1991,183(4):542-554
Experiments were carried out to determine how decreased expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) affects photosynthetic metabolism in ambient growth conditions. In a series of tobacco (Nicotiana tabacum L.) plants containing progressively smaller amounts of Rubisco the rate of photosynthesis was measured under conditions similar to those in which the plants had been grown (310 mol photons · m–2 · s–1, 350 bar CO2, 22° C). (i) There was only a marginal inhibition (6%) of photosynthesis when Rubisco was decreased to about 60% of the amount in the wildtype. The reduced amount of Rubisco was compensated for by an increase in Rubisco activation (rising from 60 to 100%), with minor contributions from an increase of its substrates (ribulose-1,5-bisphosphate and the internal CO2 concentration) and a decrease of its product (glycerate-3-phosphate). (ii) The decreased amount of Rubisco was accompanied by an increased ATP/ADP ratio that may be causally linked to the increased activation of Rubisco. An increase of highenergy-state chlorophyll fluorescence shows that thylakoid membrane energisation and high-energy-state-dependent energy dissipation at photosystem two had also increased. (iii) A further decrease of Rubisco (in the range of 50–20% of the wildtype level) resulted in a strong and proportional inhibition of CO2 assimilation. This was accompanied by a decrease of fructose-1,6-bisphosphatase activity, coupling-factor 1 (CF1)-ATP-synthase protein, NADP-malate dehydrogenase protein, and chlorophyll. The chlorophyll a/b ratio did not change, and enolase and sucrose-phosphate synthase activity did not decrease. It is argued that other photosynthetic enzymes are also decreased once Rubisco decreases to the point at which it becomes strongly limiting for photosynthesis. (iv) It is proposed that the amount of Rubisco in the wildtype represents a balance between the demands of light, water and nitrogen utilisation. The wildtype overinvests about 15% more protein in Rubisco than is needed to avoid a strict Rubisco limitation of photosynthesis. However, this excess Rubisco allows the wildtype to operate with lower thylakoid energisation, and decreased high-energy-state-dependent energy dissipation, hence increasing light-use efficiency by about 6%. It also allows the wildtype to operate with a lower internal CO2 concentration in the leaf and a lower stomatal conductance at a given rate of photosynthesis, so that instantaneous water-use efficiency is marginally (8%) increased.Abbreviations Ci
CO2 concentration in the air spaces within the leaf
- CF1
coupling factor 1
- Chl
chlorophyll Fru1
- 6bisP
fructose-1,6-bisphosphate
- Fm
fluorescence yield with a saturating pulse in dark-adapted material
- Fo
ground-level of fluorescence obtained using a weak non-actinic modulated beam in the dark
- PGA
glycerate-3-phosphate
-
rbcS
gene for the nuclear-encoded small subunit of Rubisco
- Rubisco
ribulose-1,5-bisphosphate carboxylase-oxygenase
- Ru1, 5bisP
ribulose-1,5-bisphosphate 相似文献
18.
Effects of growth-light quantity,growth-light quality and CO2 concentration on Rubisco deactivation during low PFD or darkness 总被引:4,自引:0,他引:4
The effects of CO2 concentration and the effects of growth-light conditions on Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) deactivation were examined for Spinacea oleracea (spinach). Rubisco deactivation kinetics and the degree that Rubisco activation limited the rise in photosynthesis following an increase in photon flux density (PFD) were determined from gas-exchange time courses. There were no significant differences in the apparent relaxation time for Rubisco deactivation among leaves exposed to high or low CO2 (50 or 1000 mol mol-1) and low PFD (170 mol m-2 s-1) or darkness. However, when PFD was increased to 1700 mol m-2 s-1 following a period of low PFD or darkness, leaves exposed to low CO2 × low PFD showed a lower contribution to the photosynthetic induction process by the activation of Rubisco than leaves exposed to the other treatments. For the growth-light experiments, spinach was grown under high PFD × high red:far-red ratio (R:FR), low PFD × high R:FR, or low PFD × low R:FR light environments. Leaves that matured under the low PFD × low R:FR treatment showed a lower percent change in photosynthesis due to Rubisco activation than leaves exposed to the other growth-light treatments. However, there were no significant differences among the growth-light treatments in the maximum contribution of Rubisco activation to the induction response or in the apparent relaxation time for Rubisco deactivation during shade events. 相似文献
19.
The regulation of component processes of photosynthesis in transgenic tobacco with decreased phosphoribulokinase activity 总被引:2,自引:0,他引:2
Dimah Z. Habash Martin A. J. Parry Saroj Parmar Matthew J. Paul Simon Driscoll Jacqueline Knight John C. Gray David W. Lawlor 《Photosynthesis research》1996,49(2):159-167
Tobacco plants (Nicotiana tabacum L.) transformed with an inverted cDNA encoding ribulose 5-phosphate kinase (phosphoribulokinase,PRK; EC 2.7.1.19) were employed to study the in vivo relationship between photosynthetic electron transport and the partitioning of electron transport products to major carbon metabolism sinks under conditions of elevated ATP concentrations and limited ribulose 1,5-bisphosphate (RuBP) regeneration. Simultaneous measurements of room temperature chlorophyll fluorescence and CO2 gas exchange were conducted on intact leaves. Under ambient CO2 concentrations and light intensities above those at which the plants were grown, transformants with only 5% of PRK activity showed down-regulation of PS II activity and electron transport in response to a decrease in net carbon assimilation when compared to wild-type. This was manifested as a decline in the efficiency of PS II electron transport (PS II), an increase in dissipation of excess absorbed light in the antennae of PS II and a decline in: total linear electron transport (J1), electron transport dedicated to carbon assimilation (JA) and electron transport allocated to photorespiration (JL). The transformants showed no alteration in the Rubisco specificity factor measured in vitro and calculated in vivo but had a relatively smaller ratio of RuBP oxygenation to carboxylation rates (vo/vc), due to a higher CO2 concentration at the carboxylation site (Cc). The relationship between PS II and CO
2was similar in transformants and wild-type under photorespiratory conditions demonstrating no change in the intrinsic relationship between PS II function and carbon assimilation, however, a novel result of this study is that this similar relationship occurred at different values of quantum flux, J1, JA, JL and vo/vc in the transformant. For both wild-type and transformants, an assessment was made of the possible presence of a third major sink for electron transport products, beside RuBP oxygenation and carboxylation, the data provided no evidence for such a sink.Abbreviations Cc
CO2 concentration at the site of carboxylation
- Ci
intercellular CO2 concentration
- gm
mesophyll conductance to CO2
- J1
total linear electron flow
- JA
linear electron flow allocated to CO2 assimilation
- Jc
linear electron flow supporting carbon reduction and oxidation cycles
- JL
linear electron flow allocated to photorespiration (RuBP oxygenation and fixation of released photorespiratory CO2)
- PRK
phosphoribulokinase
- qP, qN
coefficients for photochemical and non-photochemical quenching of fluorescence respectively
- Rubisco
ribulose 1,5-bisphosphate carboxylase-oxygenase
- S
Rubisco specificity to CO2/O2
- vc, vo
rates of RuBP carboxylation and RuBP oxygenation, respectively
- CO
2
relative quantum yield of CO2 assimilation
- C
maximum CO
2 under non-photorespiratory conditions
- exc
the efficiency of excitation capture by open PS II centres
- PS II
relative quantum yield of PS II electron transport 相似文献
20.
Joanna C. Scales Martin A. J. Parry Michael E. Salvucci 《Photosynthesis research》2014,119(3):355-365
Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes carboxylation of ribulose-1,5-bisphosphate, the first in a series of reactions leading to the incorporation of atmospheric CO2 into biomass. Rubisco requires Rubisco activase (RCA), an AAA+ ATPase that reactivates Rubisco by remodelling the conformation of inhibitor-bound sites. RCA is regulated by the ratio of ADP:ATP, with the precise response potentiated by redox regulation of the alpha-isoform. Measuring the effects of ADP on the activation of Rubisco by RCA using the well-established photometric assay is problematic because of the adenine nucleotide requirement of 3-phosphoglycerate (3-PGA) kinase. Described here is a novel assay for measuring RCA activity in the presence of variable ratios of ADP:ATP. The assay couples the formation of 3-PGA from ribulose 1,5-bisphosphate and CO2 to NADH oxidation through cofactor-dependent phosphoglycerate mutase, enolase, PEP carboxylase and malate dehydrogenase. The assay was used to determine the effects of Rubisco and RCA concentration and ADP:ATP ratio on RCA activity, and to measure the activation of a modified Rubisco by RCA. Variations of the basic assay were used to measure the activation state of Rubisco in leaf extracts and the activity of purified Rubisco. The assay can be automated for high-throughput processing by conducting the reactions in two stages. 相似文献